Carbamate-substituted pyrazolopyridine-derivatives

ABSTRACT

This invention relates to compounds of the formula 
                 
 
wherein R 1  is H or a di-C 1-6 -alkylaminocarbonyl raical, and R 2  is a radical of the formula —O—C(X)—NR 3 R 4  wherein X is O or S. A process for preparing such compounds, pharmaceutical compositions containing them, and methods for using them in treatment of hypertension are also disclosed and claimed.

This application is a 371 of PCT/EP01/12966 filed Nov. 9, 2001.

The present invention relates to novel chemical compounds whichstimulate soluble guanylate cyclase, to the preparation thereof and tothe use thereof as medicaments, in particular as medicaments for thetreatment of cardiovascular disorders.

One of the most important cellular transmission systems in mammaliancells is cyclic guanosine monoposphate (cGMP). Together with nitricoxide (NO), which is released from the endothelium and transmitshormonal and mechanical signals, it forms the NO/cGMP system. Guanylatecyclases catalyse the biosynthesis of cGMP from guanosine triposphate(GTP). The representatives of this family disclosed to date can bedivided both according to structural features and according to the typeof ligands into two groups: the particulate guanylate cyclases which canbe stimulated by natriuretic peptides, and the soluble guanylatecyclases which can be stimulated by NO. The soluble guanylate cyclasesconsist of two subunits and very probably contain one heme perheterodimer, which is part of the regulatory site. The latter is ofcentral importance for the mechanism of activation. NO is able to bindto the iron atom of heme and thus markedly increase the activity of theenzyme. Heme-free preparations cannot, by contrast, be stimulated by NO.CO is also able to attach to the central iron atom of heme, but thestimulation by CO is distinctly less than that by NO.

Through the production of cGMP and the regulation, resulting therefrom,of phosphodiesterases, ion channels and protein kinases, guanylatecyclase plays a crucial part in various physiological processes, inparticular in the relaxation and proliferation of smooth muscle cells,in platelet aggregation and adhesion and in neuronal signaltransmission, and in disorders caused by an impairment of theaforementioned processes. Under pathophysiological conditions, theNO/cGMP system may be suppressed, which may lead for example to highblood pressure, platelet activation, increased cellular proliferation,endothelial dysfunction, atherosclerosis, angina pectoris, heartfailure, thromboses, stroke and myocardial infarction.

A possible way of treating such disorders which is independent of NO andaims at influencing the cGMP signal pathway in organisms is a promisingapproach because of the high efficiency and few side effects which areto be expected.

Compounds, such as organic nitrates, whose effect is based on NO have todate been exclusively used for the therapeutic stimulation of solubleguanylate cyclase. NO is produced by bioconversion and activates solubleguanylate cyclase by attaching to the central iron atom of heme. Besidesthe side effects, the development of tolerance is one of the crucialdisadvantages of this mode of treatment.

Some substances which directly stimulate soluble guanylate cyclase, i.e.without previous release of NO, have been described in recent years,such as, for example, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole(YC-1, Wu et al., Blood 84 (1994), 4226; Mülsch et al., Brit. J.Pharmacol. 120 (1997), 681), fatty acids (Goldberg et al, J. Biol. Chem.252 (1977), 1279), diphenyliodonium hexafluorophosphate (Pettibone etal., Eur. J. Pharmacol. 116 (1985), 307), isoliquiritigenin (Yu et al.,Brit. J. Pharmacol. 114 (1995), 1587) and various substituted pyrazolederivatives (WO 98/16223).

In addition, WO 98/16507, WO 98/23619, WO 00/06567, WO 00/06568, WO00/06569 and WO 00/21954 describe pyrazolopyridine derivatives asstimulators of soluble guanylate cyclase. Also described inter alia inthese patent applications are pyrazolopyridines having a pyrimidineresidue in position 3. Compounds of this type have very high in vitroactivity in relation to stimulating soluble guanylate cyclase. However,it has emerged that these compounds have some disadvantages in respectof their in vivo properties such as, for example, their behavior in theliver, their pharmacokinetic behavior, their dose-response relation ortheir metabolic pathway.

It was therefore the object of the present invention to provide furtherpyrazolopyridine derivatives which act as stimulators of solubleguanylate cyclase but do not have the disadvantages, detailed above, ofthe compounds from the prior art.

This object is achieved according to the present invention by thecompounds claimed in claim 1. This new class of pyrazolopyridinederivatives is distinguished by having in position 3 a pyrimidineresidue which has a particular substitution pattern, namely a carbamateor thiocarbamate residue in position 5 of the pyrimidine ring, and anamino group or a dialkylamide group in position 4 of the pyrimidinering.

The present invention specifically relates to compounds of the formula(I)

in which

-   R¹ is hydrogen or a di-C₁₋₆-alkylaminocarbonyl radical,-   R² is a radical of the formula —O—CX—NR³R⁴,    -   where    -   X is O or S;    -   R³ and R⁴ may be identical or different from one another and is        a radical from the group consisting of H, optionally substituted        C₁₋₆-alkyl, optionally substituted C₁₋₆-alkoxy-C₁₋₆-alkyl,        optionally substituted hydroxy-C₁₋₆-alkyl, optionally        substituted C₂₋₆-alkenyl, optionally substituted        C₁₋₆-alkylcarbonyloxy-C₁₋₆-alkyl, optionally substituted        hydroxycarbonyl-C₁₋₆-alkyl, phenyl which is optionally        substituted by a C₁₋₆-alkyl radical, or a saturated five- to        seven-membered heterocycle which is optionally linked via a        C₁₋₆-alkyl radical to the nitrogen atom, or optionally        substituted C₃₋₈-cycloalkyl, where R³ and R⁴ cannot        simultaneously be H;        -   or        -   R³ and R⁴ together with the nitrogen atom to which they are            bonded form a five- to seven-membered saturated heterocycle            which may optionally contain a further heteroatom from the            group of N, O, S and/or may optionally be substituted or            fused to a phenyl ring;            and salts, isomers and hydrates thereof.

Preference is given according to the present invention to compounds ofthe formula (I) in which

-   R¹ is hydrogen or a di-C₁₋₆-alkylaminocarbonyl radical,-   R² is a radical of the formula —O—CX—NR³R⁴,    -   where    -   X is O or S;    -   R³ and R⁴ may be identical or different and is a radical from        the group consisting of H, C₁₋₆-alkyl which optionally has a CN        or a halogen substituent, C₁₋₆-alkoxy-C₁₋₆-alkyl,        hydroxy-C₁₋₆-alkyl, C₂₋₆-alkenyl,        C₁₋₆-alkylcarbonyloxy-C₁₋₆-alkyl,        C₁₋₆-alkoxycarbonyl-C₁₋₆-alkyl, hydroxycarbonyl-C₁₋₆-alkyl,        phenyl, p-tolyl, a saturated five- to seven-membered heterocycle        which is linked via a C₁₋₆-alkanediyl radical to the nitrogen        atom and has up to 2 oxygen atoms, or optionally substituted        C₃₋₈-cycloalkyl, where R³ and R⁴ cannot simultaneously be H;        -   or        -   R³ and R⁴ together with the nitrogen atom to which they are            bonded form a five- to seven-membered saturated heterocycle            which may optionally contain a further heteroatom from the            group of N, O, S and/or may optionally be substituted or            fused to a phenyl ring;            and salts, isomers and hydrates thereof.

Particular preference is given in this connection to compounds of theformula (I) in which

-   R¹ is hydrogen or a diethylaminocarbonyl radical,-   R² is a radical of the formula —O—CX—NR³R⁴,    -   where    -   X is O or S;    -   R³ and R⁴ may be identical or different and is a radical from        the group consisting of H, methyl, ethyl, isopropyl, butan-2-yl,        methoxyethyl, 2-methoxy-1-methylethyl, 1-cyano-1-methylethyl,        2-cyanoethyl, 2-chloroethyl, ethoxycarbonylmethyl,        hydroxycarbonylmethyl, 2-propenyl, phenyl, p-tolyl,        1,3-dioxolan-2-methyl, cyclohexyl or cyclopentyl, where R³ and        R⁴ cannot simultaneously be H;        -   or    -   R³ and R⁴ together with the nitrogen atom to which they are        bonded form a five- or six-membered saturated heterocycle which        may optionally contain a further heteroatom from the group of N,        O and/or may optionally have a substituent from the group of        methylcarbonyl, ethoxycarbonyl or t-butoxycarbonyl, or together        are 1,2,3,4-tetrahydroquinolin-N-yl;        and salts, isomers and hydrates thereof.

The compounds of the invention of the general formula (I) may also be inthe form of their salts. Mention may generally be made here of saltswith organic or inorganic bases or acids.

Physiologically acceptable salts are preferred for the purposes of thepresent invention. Physiologically acceptable salts of the compoundsaccording to the invention may be salts of the substances according tothe invention with mineral acids, carboxylic acids or sulfonic acids.Particularly preferred examples are salts with hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid,naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid,tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.

Physiologically acceptable salts may likewise be metal or ammonium saltsof the compounds according to the invention having a free carboxylgroup. Particularly preferred examples are sodium, potassium, magnesiumor calcium salts, and ammonium salts derived from ammonia or organicamines such as, for example, ethylamine, di- or triethylamine, di- ortriethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine,lysine or ethylenediamine.

The compounds of the invention may exist in stereoisomeric forms whicheither are related as image and mirror image (enantiomers) or which arenot related as image and mirror image (diastereomers). The inventionrelates both to the enantiomers or diastereomers and to respectivemixtures thereof. The racemic forms can, just like the diastereomers, beseparated into the stereoisomerically pure constituents in a knownmanner, for example by chromatographic separation. Double bonds presentin the compounds of the invention may be in the cis or transconfiguration (Z or E form).

A further possibility is for certain compounds to exist in tautomericforms. This is known to the skilled person, and the invention likewiseencompasses such compounds.

The compounds of the invention may also occur in the form of theirhydrates, where the number of water molecules bound to the moleculedepends on the particular compound of the invention.

Unless indicated otherwise, substituents generally have the followingmeaning for the purposes of the present invention:

Alkyl is generally a straight-chain or branched hydrocarbon radicalhaving 1 to 20 carbon atoms. Examples which may be mentioned are methyl,ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl,isohexyl, heptyl, isoheptyl, octyl and isooctyl, nonyl, decyl, dodeyl,eicosyl.

Alkylene is generally a straight-chain or branched hydrocarbon bridgehaving 1 to 20 carbon atoms. Examples which may be mentioned aremethylene, ethylene, propylene, α-methylethylene, β-methylethylene,α-ethylethylene, β-ethylethylene, butylene, α-methylpropylene,β-methylpropylene, γ-methylpropylene, α-ethylpropylene,β-ethylpropylene, γ-ethylpropylene, pentylene, hexylene, heptylene,octylene, nonylene, decylene, dodeylene and eicosylene.

Alkenyl is generally a straight-chain or branched hydrocarbon radicalhaving 2 to 20 carbon atoms and one or more, preferably having one ortwo, double bonds. Examples which may be mentioned are allyl, propenyl,isopropenyl, butenyl, isobutenyl, pentenyl, isopentenyl, hexenyl,isohexenyl, heptenyl, isoheptenyl, octenyl, isooctenyl.

Alkynyl is generally a straight-chain or branched hydrocarbon radicalhaving 2 to 20 carbon atoms and one or more, preferably having one ortwo, triple bonds. Examples which may be named are ethynyl, 2-butynyl,2-pentynyl and 2-hexynyl.

Acyl is generally straight-chain or branched lower alkyl having 1 to 9carbon atoms which is linked via a carbonyl group. Examples which may bementioned are: acetyl, ethylcarbonyl, propylcarbonyl, isopropylcarbonyl,butylcarbonyl and isobutylcarbonyl.

Alkoxy is generally a straight-chain or branched hydrocarbon radicalhaving 1 to 14 carbon atoms which is linked via an oxygen atom. Exampleswhich may be mentioned are methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, pentoxy isopentoxy, hexoxy, isohexoxy, heptoxy, isoheptoxy,octoxy or isooctoxy. The terms “alkoxy” and “alkyloxy” are usedsynonymously.

Alkoxyalkyl is generally an alkyl radical having up to 8 carbon atomswhich is substituted by an alkoxy radical having up to 8 carbon atoms.

Alkoxycarbonyl may be represented for example by the formula

In this case, alkyl is generally a straight-chain or branchedhydrocarbon radical having 1 to 13 carbon atoms. Examples which may bementioned are the following alkoxycarbonyl radicals: methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl orisobutoxycarbonyl.

Cycloalkyl is generally a cyclic hydrocarbon radical having 3 to 8carbon atoms. Cyclopropyl, cyclopentyl and cyclohexyl are preferred.Examples which may be mentioned are cyclopentyl, cyclohexyl, cycloheptyland cyclooctyl.

Cycloalkoxy is for the purposes of the invention an alkoxy radical whosehydrocarbon radical is a cycloalkyl radical. The cycloalkyl radicalgenerally has up to 8 carbon atoms. Examples which may be mentioned are:cyclopropyloxy and cyclohexyloxyl. The terms “cycloalkoxy” and“cycloalkyloxy” are used synonymously.

Aryl is generally an aromatic radical having 6 to 10 carbon atoms.Preferred aryl radicals are phenyl and naphthyl.

Halogen is for the purposes of the invention fluorine, chlorine, bromineand iodine.

Heterocycle is for the purposes of the invention in general a saturated,unsaturated or aromatic 3- to 10-membered, for example 5- or 6-membered,heterocycle which may contain up to 3 heteroatoms from the series S, Nand/or O and, in the case of a nitrogen atom, also be bonded via thelatter. Examples which may be mentioned are: oxadiazolyl, thiadiazolyl,pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thienyl, furyl,pyrrolyl, pyrrolidinyl, piperazinyl, tetrahydropyranyl,tetrahydrofuranyl, 1,2,3 triazolyl, thiazolyl, oxazolyl, imidazolyl,morpholinyl or piperidyl. Preference is given to thiazolyl, furyl,oxazolyl, pyrazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl andtetrahydropyranyl. The term “heteroaryl” (or “hetaryl”) stands for anaromatic heterocyclic radical.

The compounds of the invention of the formula (I) can be prepared byreacting the compound of the formula (II)

with the compound of the formula (III)

in an organic solvent in the presence of a base with heating andsubsequently converting the ether group into the free hydroxyl group togive compounds of the formula (IV)

and subsequently reacting with compounds of the formula X—CO—NR³R⁴in which

-   -   X is a halogen radical or alkoxy radical,    -   R³ and R⁴ have the meaning indicated above;        in an organic solvent, where appropriate in the presence of a        base, and where appropriate with subsequent removal of        protective groups to give compounds of the formula (I).

The compound of the formula (II) can be prepared as shown in thefollowing reaction scheme:

The compound of the formula (II) can be obtained in a multistagesynthesis from the sodium salt of ethyl cyanopyruvate which is knownfrom the literature (Borsche and Manteuffel, Liebigs. Ann. Chem. 1934,512, 97). Reaction thereof with 2-fluorobenzylhydrazine with heatingunder a protective gas atmosphere in an inert solvent such as dioxaneresults in ethyl 5-amino-1-(2-fluorobenzyl)pyrazole-3-carboxylate, whichcyclizes to the corresponding pyridine derivative by reaction withdimethylaminoacrolein or 1,1,3,3-tetramethoxypropane in acidic mediumwith heating under a protective gas atmosphere. This pyridine derivativeethyl 1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxylate isconverted by a multistage sequence consisting of conversion of the esterwith ammonia into the corresponding amide, dehydration with adehydrating agent such as trifluoacetic anhydride to the correspondingnitrile derivative, reaction of the nitrile derivative with sodiumethoxide and finally reaction with ammonium chloride into the compoundof the formula (II).

The compound of the formula (III) can be prepared from the compounds,which can be purchased (e.g. from Aldrich),t-butoxybis(dimethylamino)methane and methoxyacetonitrile by reactingthese reactants preferably in equimolar quantities, where appropriate inan organic inert solvent such as a cyclic ether, preferably dioxane,preferably under atmospheric pressure and stirring the reaction solutionfor several hours, for example 12 hours, at elevated temperature, forexample 60-110° C., preferably 70-90° C., in particular 80° C.

Reaction of the compounds of the formulae (II) and (III) to give thecompound of the formula (IV) can be carried out by employing thereactants in equimolar quantities or by using the compound of theformula (III) in slight excess in an organic solvent, for example analcohol, preferably isoamyl alcohol in the presence of a small quantityof a base, for example an organic amine, in particular piperidine,preferably under atmospheric pressure and stirring the reaction solutionfor several hours, for example 12 hours, at elevated temperature, forexample 60-130° C., preferably 80-120° C., in particular 110° C., andsubsequently liberating the hydroxyl group by reacting the compoundobtained in this way with a preferably equimolar quantity of a thiolsuch as, for example, thiophenol in the presence of a small quantity ofa base such as an alkali metal base, for example an alkali metalcarbonate, preferably potassium carbonate in an organic solvent such as,for example, 1-methyl-2-pyrrolidone, preferably under atmosphericpressure and stirring the reaction solution for some hours, for example1 hour, at elevated temperature, for example 100-200° C., preferably150-200° C.

The compounds of the formula (IV) can be reacted with compounds of theformula X—CONR³R⁴ where X, R³ and R⁴ are as defined above to give thecompounds of the invention of the formula (I). These carbamoylderivatives can either be purchased or be obtained in a manner known tothe skilled worker. The reaction of appropriate secondary amines withphosgene or phosgene substitutes such as trichloromethyl chloroformate(diphosgene) or bis(trichloromethyl) carbonate (triphosgene) may bementioned here by way of example (cf. J. March, Advanced OrganicSynthesis, 3^(rd) ed., Wiley 1985, 370, D. Hoppe, Synthesis 1996,149-154). The secondary amines required for this can either be purchasedor be obtained in a manner known to the skilled worker, for example byreacting a primary amine with an appropriate aldehyde or ketone using areducing agent conventionally employed for such reactions, for example ametal hydride complex, preferably an alkali metal hydride complex suchas sodium cyanoborohydride (“Reductive Amination”, cf. K.-L. Yu, J.Ostrowski, P. Reczek, M. M. Mansuri, J. E. Starrett Jr., SyntheticCommunications, 1995, 25, 2819-2827).

Reaction of the compounds of the formula (IV) with compounds of theformula X—CONR³R⁴ where X, R³ and R⁴ are as defined above to give thecompounds of the invention of the formula (I) preferably takes placeeither in an organic solvent conventionally used for such reactions,such as, for example, a cyclic ether, in particular tetrahydrofuran(THF), in the presence of an equimolar quantity or of a slight excess ofa base, with preference an alkali metal base, preferably of an alkalimetal hydride and particularly preferably sodium hydride, preferablyunder atmospheric pressure and stirring the reaction solution forseveral hours, for example 12 hours, at room temperature. The reactantsare in this case employed in equimolar quantities or the carbamoylderivative in slight excess. Another possibility is to carry out thereaction in pyridine without another base, preferably under atmosphericpressure and stirring the reaction solution for some hours, for example2 hours, at elevated temperature of 60 to 130° C., preferably 80 to 120°C. and in particular at 110° C.

Reaction of the compound of the formula (IV) with thiocarbamoylchlorides to give the corresponding carbamates can also take placeanalogously. Reaction in pyridine is preferred. The thiocarbamoylchlorides used according to the invention can be purchased.

It is subsequently possible for protective groups which are presentwhere appropriate on the molecule to be removed in a manner known to theskilled worker. Concerning this, reference may be made for example to T.W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, secondedition, New York, 1991, concerning possible protective groups and theremoval thereof from the target compound.

The compounds of the invention of the general formula (I) show avaluable range of pharmacological effects which could not have beenpredicted.

The compounds of the invention of the general formula (I) lead tovasorelaxation, inhibition of platelet aggregation and to a reduction inblood pressure and to an increase in coronary blood flow. These effectsare mediated by direct stimulation of soluble guanylate cyclase and anintracellular increase in cGMP. In addition, the compounds of theinvention of the general formula (I) enhance the effect of substanceswhich increase the cGMP level, such as, for example, EDRF (endotheliumderived relaxing factor), NO donors, protoporphyrin IX, arachidonic acidor phenylhydrazine derivatives.

They can therefore be employed in medicaments for the treatment ofcardiovascular disorders such as, for example, for the treatment of highblood pressure and heart failure, stable and unstable angina pectoris,peripheral and cardiac vascular disorders, of arrhythmias, for thetreatment of thromboembolic disorders and ischemias such as myocardialinfarction, stroke, transistorily and ischemic attacks, disturbances ofperipheral blood flow, prevention of restenoses as after thrombolysistherapies, percutaneously transluminal angioplasties (PTAs),percutaneously transluminal coronary angioplasties (PTCAs), bypass andfor the treatment of arteriosclerosis, asthmatic disorders and diseasesof the urogenital system such as, for example, prostate hypertrophy,erectile dysfunction, female sexual dysfunction, osteoporosis,gastroparesis and incontinence.

The compounds of the present invention of the general formula (I) arealso active substances for controlling central nervous system diseasescharacterized by disturbances of the NO/cGMP system. They are suitablein particular for improving perception, concentration, learning ormemory after cognitive impairments like those occurring in particular inassociation with situations/diseases/syndromes such as mild cognitiveimpairment, age-associated learning and memory impairments,age-associated memory loss, vascular dementia, craniocerebral trauma,stroke, dementia occurring after strokes (post stroke dementia),post-traumatic brain trauma, general concentration impairments,concentration impairments in children with learning and memory problems,Alzheimer's disease, vascular dementia, Lewy body dementia, dementiawith degeneration of the frontal lobes including Pick's syndrome,Parkinson's disease, progressive nuclear palsy, dementia withcorticobasal degeneration, amyolateral sclerosis (ALS), Huntington'sdisease, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacobdementia, HIV dementia, schizophrenia with dementia or Korsakoff'spsychosis. They are also suitable for the treatment of central nervoussystem disorders such as states of anxiety, tension and depression,CNS-related sexual dysfunctions and sleep disturbances, and forcontrolling pathological disturbances of the intake of food, stimulantsand addictive substances.

The active ingredients are furthermore also suitable for controllingcerebral blood flow and thus represent effective agents for controllingmigraines.

They are also suitable for the prophylaxis and control of the sequelaeof cerebral infarctions such as stroke, cerebral ischemias andcraniocerebral trauma. The compounds of the invention of the generalformula (I) can likewise be employed for controlling states of pain.

In addition, the compounds of the invention have an anti-inflammatoryeffect and can therefore be employed as anti-inflammatory agents.

Furthermore, the present invention also encompasses the combination ofthe compounds of the invention of the general formula (I) with organicnitrates or NO donors.

Organic nitrates and NO donors for the purposes of the invention aregenerally substances which display their therapeutic effect via releaseof NO or NO species. Sodium nitroprusside, nitroglycerin, isosorbidedinitrate, isosorbide mononitrate, molsidomine and SIN-1 are preferred.

In addition, the present invention also encompasses the combination withcompounds which inhibit breakdown of cyclic guanosine monophosphate(cGMP). These are in particular inhibitors of phosphodiesterases 1, 2and 5; nomenclature of Beavo and Reifsnyder (1990), TiPS 11 pp. 150 to155. These inhibitors potentiate the effect of the compounds of theinvention, and increase the desired pharmacological effect.

Biological Investigations Vasorelaxant Effect In Vitro

Rabbits are stunned by a blow to the back of the neck and areexsanguinated. The aorta is removed, freed of adherent tissue anddivided into rings 1.5 mm wide, which are put singly under tension in 5ml organ baths containing carbogen-gassed Krebs-Henseleit solution at37° C. with the following composition (mM): NaCl: 119; KCl: 4.8; CaCl₂×2H₂O: 1; MgSO₄×7 H₂O: 1.4; KH₂PO₄: 1.2; NaHCO₃: 25; glucose: 10. Theforce of contraction is detected with Statham UC2 cells, amplified anddigitized via A/D converters (DAS-1802 HC, Keithley Instruments Munich)and recorded in parallel on chart recorders. A contraction is generatedby adding phenylephrine to the bath cumulatively in increasingconcentration. After several control cycles, the substance to beinvestigated is investigated in each further run in increasing dosage ineach case, and the height of the contraction is compared with the heightof the contraction reached in the last preceding run. The concentrationnecessary to reduce the height of the control value by 50% (IC₅₀) iscalculated from this. The standard application volume is 5 μl, and theDMSO content in the bath solution corresponds to 0.1%. The results arelisted in Table 1 below:

TABLE 1 Vasorelaxant effect in vitro Example no. IC₅₀ [μM] 2 0.65 6 0.2711 0.52 15 0.32 19 0.42 26 0.34

Determination of the Liver Clearance In Vitro

Rats are anesthetized and heparinized, and the liver is perfused in situvia the portal vein. Primary rat hepatocytes are then obtained ex vivofrom the liver using collagenase solution. 2·10⁶ hepatocytes per ml wereincubated with in each case the same concentration of the compound to beinvestigated at 37° C. The decrease in the substrate to be investigatedover time was determined bioanalytically (HPLC/UV, HPLC/fluorescence orLC/MSMS) at 5 time points in each case in the period 0-15 min after thestart of incubation. The clearance was calculated therefrom via thenumber of cells and the weight of the liver.

Determination of the Plasma Clearance In Vivo

The substance to be investigated is administered intravenously assolution to rats via the tail vein. Blood is taken from the rats atfixed times and is heparinized, and plasma is obtained therefrom byconventional procedures. The substance is quantified in the plasmabioanalytically. The pharmacokinetic parameters are calculated from theplasma concentration/time courses found in this way via conventionalnon-compartmental methods used for this purpose.

The present invention includes pharmaceutical preparations which,besides non-toxic, inert pharmaceutically suitable carriers, comprisethe compounds of the invention of the general formula (I), and processfor the production of these preparations.

The active ingredient may, where appropriate, also be present inmicroencapsulated form in one or more of the carriers indicated above.

The therapeutically effective compounds of the general formula (I)should be present in the pharmaceutical preparations mentioned above ina concentration of about 0.1 to 99.5, preferably of about 0.5 to 95, %by weight of the complete mixture.

The pharmaceutical preparations mentioned above may, apart from thecompounds of the invention of the general formula (I), also compriseother active pharmaceutical ingredients.

It has generally proved advantageous both in human and in veterinarymedicine to administer the active ingredient(s) of the invention intotal amounts of about 0.01 to about 700, preferably 0.01 to 100, mg/kgof body weight per 24 hours, where appropriate in the form of aplurality of single doses, to achieve the desired results. A single dosepreferably contains the active ingredients of the invention in amountsof about 0.1 to about 80, in particular 0.1 to 30, mg/kg of body weight.

The present invention is explained in more detail below by means ofnon-restrictive preferred examples. Unless indicated elsewhere, allquantitative data relate to percentages by weight.

EXAMPLES

Abbreviations:

-   RT: Room temperature-   EA: Ethyl acetate-   MCPBA: m-Chloroperoxybenzoic acid-   BABA: n-Butyl acetate/n-butanol/glacial acetic acid/phosphate buffer    pH 6 (50:9:25.15; org. phase)-   DMF: N,N-Dimethylformamide    Mobile phases for thin-layer chromatography:-   T1 E1: Toluene/ethyl acetate (1:1)-   T1 EtOH1: Toluene methanol (1:1)-   C1 E1: Cyclohexane/ethyl acetate (1:1)-   C1 E2: Cyclohexane/ethyl acetate (1:2)    Methods for determining the HPLC retention times:    Method A (HPLC-MS):-   Eluent: A=CH₃CN B=0.6 g 30% HCl/l H₂O-   Flow rate: 0.6 ml/min-   Column oven: 50° C.-   Column: Symmetry C18 2.1*150 mm-   Gradient:

Time (min) % A % B Flow rate (ml/min) 0 10 90 0.6 4 90 10 0.6 9 90 100.8Method B (HPLC):

-   Eluent: A=5 ml HClO₄/l H₂O, B=CH₃CN-   Flow rate: 0.75 ml/min-   L-R temperature: 30.00° C. 29.99° C.-   Column: Kromasil C18 60*2 mm-   Gradient:

Time (min) % A % B 0.50 98 2 4.50 10 90 6.50 10 90 6.70 98 2 7.50 98 2Method C (HPLC):

-   Eluent: A=H₃PO₄ 0.01 mol/l, B=CH₃CN-   Flow rate: 0.75 ml/min-   L-R temperature: 30.01° C. 29.98° C.-   Column: Kromasil C18 60*2 mm

Gradient: Time (min) % A % B 0.00 90 10 0.50 90 10 4.50 10 90 8.00 10 908.50 90 10 10.00 90 10Method D (chiral HPLC):

-   Eluent: 50% isohexane, 50% ethanol-   Flow rate: 1.00 ml/min-   Temperature: 40° C.-   Column: 250*4.6 mm, packed with Chiralcel OD, 10 μm    Method E (HPLC-MS):-   Eluent: A=CH₃CN B=0.3 g 30% HCl/l H₂O-   Flow rate: 0.9 ml/min-   Column oven: 50° C.-   Column: Symmetry C18 2.1*150 mm-   Gradient:

Time (min) % A % B Flow rate (ml/min) 0 10 90 0.9 3 90 10 1.2 6 90 101.2Method F:

-   Eluent: A=CH₃CN+0.1% HCOOH B=H₂O+0.1% HCOOH-   Column oven: 40° C.-   Column: Symmetry C18 2.1*150 mm-   Gradient:

Time (min) % A % B Flow rate (ml/min) 0 10 90 0.5 4 90 10 0.5 6 90 100.5 6.1 10 90 1.0 7.5 10 90 0.5

Starting Compounds I. Synthesis of3,3-bis(dimethylamino)-2-methoxypropionitrile

40.0 g (229.5 mmol) of ter-butoxybis(dimethylamino)methane and 16.3 g(229.5 mmol) of methoxyacetonitrile are stirred at 80° C. overnight. Forworkup, volatile material is stripped off in a rotary evaporator, andthe residue is distilled in a kugelrohr under high vacuum at 140° C. TheNMR spectrum (300 MHz, D₆-DMSO) shows that the product contains theenamine as E/Z mixture resulting from elimination of dimethylamine. Theproduct mixture is employed in the next reaction without furtherpurification.

Yield: 24.7 g (60%)

II. Synthesis of1-(2-fluorobenzyl)1H-pyrazolo[3,4-b]pyridine-3-carboxamidine 2A) Ethyl5-amino-1-(2-fluorobenzyl)pyrazol-3-carboxylate

111.75 g (75 ml, 0.98 mol) of trifluoroacetic acid are added to 100 g(0.613 mmol) of the sodium salt of ethyl cyanopyruvate (preparation inanalogy to Borsche and Manteuffel, Liebigs Ann. 1934, 512, 97) in 2.5 lof dioxane under argon at room temperature with efficient stirring, andthe mixture is stirred for 10 min, during which most of the precursordissolves. Then 85.93 g (0.613 mol) of 2-fluorobenzylhydrazine are addedand the mixture is boiled overnight. After cooling, the sodiumtrifluoroacetate crystals which have separated out are filtered off withsuction and washed with dioxane, and the crude solution is reactedfurther.

2B) Ethyl 1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxylate

The solution obtained from 2A) is mixed with 61.25 ml (60.77 g, 0.613mmol) of dimethylaminoacrolein and 56.28 ml (83.88 g, 0.736 mol) oftrifluoroacetic acid and boiled under argon for 3 days. The solvent isthen evaporated in vacuo, and the residue is added to 2 l of water andextracted three times with 1 l of ethyl acetate each time. The combinedorganic phases are dried over magnesium sulfate and concentrated in arotary evaporator. Chromatography is carried out on 2.5 kg of silicagel, eluting with a toluene/toluene-ethyl acetate=4:1 gradient. Yield:91.6 g (49.9% of theory over two stages).

Melting point 85° C.

R_(f) (SiO₂, T1E1): 0.83

2C) 1-(2-Fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide

10.18 g (34 mmol) of the ester obtained in example 2B) are introducedinto 150 ml of methanol saturated with ammonia at 0-10° C. Stirring atroom temperature for two days is followed by concentration in vacuo.

R_(f) (SiO₂, T1E1): 0.33

2D) 3-Cyano-1-(2-fluorobenyl)-1H-pyrazolo[3,4-b]pyridine

36.1 g (133 mmol) of1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide from example2C) are dissolved in 330 ml of THF, and 27 g (341 mmol) of pyridine areadded. Then, over the course of 10 min, 47.76 ml (71.66 g, 341 mmol) oftrifluoroacetic anhydride are added, during which the temperature risesto 40° C. The mixture is stirred at room temperature overnight. It isthen added to 1 l of water and extracted three times with 0.5 l of ethylacetate each time. The organic phase is washed with saturated sodiumbicarbonate solution and with 1 N HCl, dried with MgSO4 and concentratedin a rotary evaporator.

Yield: 33.7 g (100% of theory)

Melting point: 81° C.

R_(f) (SiO₂, T1E1): 0.74

2E) Methyl (2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboximidate

30.37 g (562 mmol) of sodium methoxide are dissolved in 1.5 l ofmethanol, and 36.45 g (144.5 mmol) of3-cyano-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (from example 2D)are added. The solution obtained after stirring at room temperature for2 hours is employed directly for the next stage.

2F) 1-(2-Fluorobenzyl)1H-pyrazolo[3,4-b]pyridine-3-carboxamidine

33.76 g (32.19 ml, 562 mmol) of glacial acetic acid and 9.28 g (173mmol) of ammonium chloride are added to the solution of methyl(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboximidate in methanolobtained from example 2E), and the mixture is stirred under refluxovernight. The solvent is evaporated in vacuo, the residue is thoroughlytriturated with acetone, and the precipitated solid is filtered off withsuction.

¹H-NMR (d₆-DMSO, 200 MHz): δ=5.93 (s, 2H); 7.1-7.5 (m, 4 H); 7.55 (dd,1H); 8.12 (dd, 1H); 8.30 (dd, 1H); 9.5 (bs, 4H-exchangeable) ppm.

ME (EI): m/z=270.2 (M—HCl)

III. Synthesis of2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methoxy-4-pyrimidinylamine

46.8 g (134.8 mmol) of1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboximidamide fromexample II are dissolved in isoamyl alcohol. To this are added 24.7 g(144.2 mmol) of 3,3-bis(dimethylamino)-2-methoxypropionitrile fromexample I and 1.15 g (1.33 ml, 13.5 mmol) of piperidine, and the mixtureis left to stir at 110° C. for 3 days. For workup, it is cooled to 0°C., and the precipitated product is filtered off with suction,thoroughly washed with cold diethyl ether and dried in a vacuum oven at50° C.

Yield: 25.4 g (52.7%)

R_(f): 0.34 (dichloromethane/methanol 20:1)

¹H-NMR: (400 MHz, D₆-DMSO), δ=3.89 (2, 3H, OCH₃), 5.79 (s, 2H, CH₂),6.93 (br. S, 2H, NH₂), 7.10-7.26 (m, 3H, Ar—H), 7.31-7.39 (m, 2H, Ar—H),

7.98 (s, 1H, pyrimidine-H), 8.61 (dd, 1H, pyridine-H), 8.92 (dd, 1H,pyridine-H)

ME (EI): (ESI pos.), m/z=350.9 ([M+H]⁺), 700.8 ([2M+H]⁺)

IV. Synthesis of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinol

25.3 g (72.2 mmol) of2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methoxy4-pyrimidinylaminefrom example III are dissolved in 500 ml of 1-methyl-2-pyrrolidone. Tothis are added 7.96 g (7.42 ml, 72.2 mmol) of thiophenol and 2.50 g(18.1 mmol) of potassium carbonate, and the mixture is left to stir at190° C. for about 1 h. For workup, the solvent is condensed off, and theresidue is mixed with half-conc. ammonium chloride solution andextracted three times with ethyl acetate. Most of the productprecipitates during this. It is filtered off with suction and dried in avacuum oven at 50° C.

Yield: 18.1 g (72.3%)

R_(f): 0.44 (dichloromethane/methanol 10:1)

¹H-NMR: (300 MHz, D₆-DMSO), δ=5.78 (s, 2H, CH₂), 6.66 (br. S, 2H, NH₂),7.09-7.38 (m, 5H, Ar—H), 7.82 (s, 1H, pyrimidine-H), 8.60 (dd, 1H,pyridine-H), 8.92 (dd, 1H, pyridine-H), 9.4-10.2 (br. S, 1H, OH)

MS: (ESI pos.), m/z=337.3 ([M+H]⁺), 673.3 ([2M+H]⁺)

V. Synthesis of 3,4-dimethoxybenzyl(methyl)carbamoyl chloride

1.00 g (5.52 mmol) of 3,4-dimethoxybenzyl-N-methylamine (obtainable byreductive amination from 3,4-dimethoxybenzaldehyde) are dissolved in 20ml of anhydrous pyridine. 0.60 g (0.37 mmol, 3.04 mmol) oftrichloromethyl chloroformate (“diphosgene”) is added, and the mixtureis left to stir at room temperature overnight. The solution is employedwithout workup directly in the next stage.

VI.4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl]3,4-dimethoxybenzyl(methyl)carbamate

1.38 g (4.10 mmol) of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinolfrom example IV are suspended in 100 ml of anhydrous pyridine and heatedto 110° C. To this is added a solution of 1.20 g (4.92 mmol) of3,4-dimethoxybenzyl(methyl)carbamoyl chloride from example V in 20 ml ofanhydrous pyridine (from the reaction described above) and the mixtureis left to stir at 110° C. for about 5 h. For workup, the reactionsolution is concentrated in a rotary evaporator, and the residue ischromatographed on silica gel with dichloromethane/methanol (initially100:1, then 50:1). The product contains, according to the NMR spectrum,an unknown impurity and is employed without further purification.

Rf: 0.90 (dichloromethane/methanol 10:1)

VII. Synthesis of allyl(cyclopentyl)carbamoyl chloride

790 mg (0.48 ml, 3.99 mmol) of trichloromethylformate (“diphosgene”) aredissolved in 15 ml of dichloromethane and cooled to 0° C. To this areslowly added dropwise 1.00 g (1.17 ml, 7.986 mmol) ofallyl(cyclopentyl)amine and 1.21 g (1.67 ml, 12.0 mmol) oftriethylamine, and the mixture is left to stir at room temperatureovernight. For workup, the reaction solution is poured into ice-water,extracted three times with dichloromethane, dried over MgSO₄ andconcentrated. The crude product is employed directly in the next stage.

MS: (El), m/z (%)=187 (12, [M]⁺, Cl), 152 (30, [M—Cl]⁺), 120 (45), 69(50), 41(100)

VIII. 4-Methoxybenzyl(2-methoxyethyl)carbamoyl chloride

0.51 g (0.31 ml, 2.56 mmol) of trichloromethyl chloroformate(“diphosgene”) are dissolved in 15 ml of dichloromethane, and 1.00 g(5.12 mmol) of 4-methoxybenzyl(2-methoxyethyl)amine and 0.78 g (7.68mmol) of triethylamine are slowly added. After stirring at roomtemperature overnight, the mixture is poured into ice-water andextracted three times with dichloromethane, and the organic phase isdried over magnesium sulfate and concentrated to dryness in a rotaryevaporator. The crude product is employed without workup directly in thenext stage.

IX.4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl4-methoxybenzyl(2-methoxyethyl)carbamate

100 mg (0.30 mmol) of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinolfrom example IV are suspended in 15 ml of anhydrous pyridine and heatedto 110° C. To this are added 92.0 mg (0.36 mmol) of4-methoxybenzyl(2-methoxyethyl)carbamoyl chloride from example VIII, andthe mixture is left to stir at 110° C. for about 2 h. For workup, thereaction solution is concentrated in a rotary evaporator, the residue istaken up in dichloromethane and washed with saturated ammonium chloridesolution and with saturated sodium chloride solution, and the organicphase is separated off and concentrated in a rotary evaporator.Chromatography of the residue on silica gel withdichloromethane/methanol (100:1) afforded the product still in 57%purity and was employed without further purification in the nextreaction.

Rf: 0.86 (dichloromethane/methanol 10:1)

EXAMPLES

The carbamates were prepared using sodium hydride in tetrahydrofuran(THF) or without other base in pyridine as solvent. The illustratedexamples are given below for each of the two reaction routes.

1.4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl1-pyrrolidinecarboxylate

3.5 mg (0.15 mmol) of sodium hydride were added to a suspension of 50.0mg (0.15 mmol) of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinolfrom example IV in 2 ml of THF at RT. After the mixture had been stirredat RT for 30 min, 21.8 mg (0.16 mmol) of 1-pyrrolidinecarbonyl chloridewere added and the mixture was stirred at RT overnight. The product waspurified by thin-layer chromatography (silica gel, CH₂Cl₂/MeOH 10:1).

Yield: 50.4 g (78.2%)

¹H-NMR: (400 MHz, DMSO-d₆): δ=1.81-1.93 (m, 4H), 3.22 (t, J=6.5 Hz, 2H),3.57 t, J=6.5 Hz, 2H), 5.81 (s, 2H), 7.05-7.30 (m, 5H), 7.31-7.44 (m,2H), 8.12 (s, 1H), 8.63 (d, J=4.4 Hz, 1H), 8.92 (d, J=7.9 Hz, 1H).

MS: ESI pos.), m/z=434.4 ([M+H]⁺)

The following were obtained in the same way:

Ex. Formula Yield (%) 1H-NMR 2 (from IV and 4- morpholinecarbonylchloride)

85 1H-NMR: (300 MHz, DMSO-d₆): δ = 3.37-3.77(m, 8H), 5.82(s, 2H),7.05-7.30(m, 5H), 7.31-7.44(m, 2H), 8.12(s, 1H), 8.64(d, J = 4.4 Hz,1H), 8.94 (d, J = 7.9 Hz, 1H).

3.4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyldiethylcarbamate

100 g (0.297 mmol) of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinolfrom example IV are suspended in 15 ml of anhydrous pyridine and heatedto 110° C. 48.4 mg (0.357 mmol) of N,N-diethylcarbamoyl chloride areadded thereto, and the mixture is stirred at 110° C. for 2 h. Thereaction solution is then concentrated in a rotary evaporator. Theresidue is chromatographed on silica gel with dichloromethane/methanol100:1.

Yield: 90.5 g (69.9%)

Rf: 0.86 (dichloromethane/methanol 10:1)

¹H-NMR: (200 MHz, D₆DMSO), δ=1.07-1.28 (m, 6H, CH₃CH₂), 3.22-3.53 (m,4H, CH₃CH₂), 5.83 (s, 2H, CH₂), 7.10-7.42 (m, 7H, Ar—H and NH₂), 8.09(s, 1H, pyrimidine-H), 8.63 (dd, 1H, pyridine-H), 8.94 (dd, 1H,pyridine-H)

MS: (ESI pos.), m/z=436.2 ([M+H]⁺), 871.0 ([2M+H]⁺)

The following was prepared analogously:

4.1-{4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl}tert-butyl-1,4 piperazinedicarboxylate

Yield: 36.4%

Rf: 0.77 (dichloromethane/methanol 10:1)

¹H-NMR: (200 MHz, D₆-DMSO). The spectrum exhibits E/Z rotamerism of thecarbamate group. δ=1.39 and 1.42 (2s, 9H, C(CH₃)₃), 3.09-3.14 (m, 1H,piperazine-H), 3.26-3.34 (m, 1H, piperazine-H), 3.36-3,48 (br.s, 6H,piperazine-H), 3.52-3.64 (br.s, 2H, piperazine-H), 5.81 (s, 2H, CH₂),7.10-7.27 (m, 5H, ArH and NH₂), 7.30-7.40 (m, 2H, Ar—H), 8.12 (s, 1H,pyrimidine-H), 8.63 (dd, 1H, pyridine-H), 8.94 (dd, 1H, pyridine-H)

MS: (ESI pos.), m/z=549.1 ([M+H]⁺)

The following were prepared analogously:

Yield Ex. Formula (%) Spectroscopic data 5 (from IV and 4- acetyl-1-piperazinecarbonyl chloride))

68 MS-ESI pos. (m/z): 491.1 [M + H]⁺ Retention time (min): 3.87 (methodB) Rf CH₂Cl₂/MeOH 10:1:0.39 6 (from IV and N- isopropyl-N- methyl-aminocarbonyl chloride)

67 MS-ESI pos. (m/z): 436.2 [M + H]⁺, 870.92 [2 M + H]⁺ Retention time(min): 4.19 (method B) Rf CH₂Cl₂/MeOH 10:1:0.91 7 (from IV and N-ethyl-N-methyl- aminocarbonyl chloride)

64 MS-ESI pos. (m/z): 422.0 [M + H]⁺, 842.9 [2 M + H]⁺ Retention time(min): 4.07 (method B) Rf CH₂ Cl₂/MeOH 10:1:0.90 8 (from IV andN-(1-cyano- 1-methylethyl)-N- methyl-amino carbonyl chloride)

35 MS-ESI pos. (m/z): 461.2 [M + H]⁺ Retention time (min): 4.19 (methodB) Rf Ch₂Cl₂/MeOH 10:1:0.84 9 (from IV and N,N- dimethyl- aminocarbonylchloride)

63 MS-ESI pos. (m/z): 408.3 [M + H]⁺, 814.9 [2 M + H]⁺ Retention time(min): 3.96 (method B) Rf CH₂Cl₂/MeOH 10:1:0.73 10 (from IV and N,N-diisopropyl- aminocarbonyl chloride)

58 MS-ESI pos. (m/z): 464 [M + H]⁺, 927 [2 M + H]⁺ Retention time (min):4.44 (method B) Rf CH₂Cl₂/MeOH 10:1: 11 (from IV and N,N- diallyl-aminocarbonyl chloride)

45 MS-ESI pos. (m/z): 460 [M + H]⁺ Retention time (min): 4.34 (method B)Rf CH₂Cl₂/MeOH 10:1: 12 (from IV and N-2- chloroethyl-N- methyl-aminocarbonyl chloride)

39 MS-ESI pos. (m/z): 456 [M + H]⁺ Retention time (min): 4.16 (method B)Rf CH₂Cl₂/MeOH 10:1: 13 (from IV and 2- ethoxycarbonyl- piperidine-N-carbonyl chloride)

21 MS-ESI pos. (m/z): 520 [M + H]⁺, 1039 [2 M + H]⁺ Retention time(min): 4.39 (method B) Rf CH₂Cl₂/MeOH 10:1: 14 (from IV and N,N-diethylthiocarbonyl chloride)

79.1 MS-ESI pos. (m/z): 452 [M + H]⁺ Retention time (min): 4.43 (methodB) 15 (from IV and N-(2- methoxy-1- methylethyl)- (ethoxycarbonyl-methyl)- aminocarbonyl chloride))

62 MS-ESI pos. (m/z): 538.2 [M + H]⁺ Retention time (min): 4.39 (methodB) 16 (from IV and N- methyl-N- cyclohexylamino- carbonyl chloride)

67.6 MS-ESI pos. (m/z): 538.2 [M + H]⁺ Retention time (min): 4.53(method B) 17 (from IV and N- ethyl-N- phenylamino- carbonyl chloride)

71.4 MS-ESI pos. (m/z): 484 [M + H]⁺ Retention time (min): 4.47 (methodB) 18 (from IV and N- methyl-N- methoxyethyl- aminocarbonyl chloride)

28.5 MS-ESI pos. (m/z): 452.3 [M + H]⁺ Retention time (min): 4.10(method B) 19 (from IV and N,N- dimethylthio- carbonyl chloride)

70.4 MS-ESI pos. (m/z): 424 [M + H]⁺ Retention time (min): 4.20 (methodB) 20 (from IV and N-2- butyl-N- ethoxycarbonyl- methylamino- carbonylchloride)

57.7 MS-ESI pos. (m/z): 522 [M + H]⁺, 1043 [2 M + H]⁺ Retention time(min): 4.47 (method B) 21 (from IV and N- methyl-N-(1,3- dioxolan-2-yl)-methylamino- carbonyl chloride)

22.2 MS-ESI pos. (m/z): 480 [M + H]⁺ Retention time (min): 4.05 (methodB) 22 (from IV and N-(p- tolyl)-N-(2- cyanoethyl)- aminocarbonylchloride)

20.4 MS-ESI pos. (m/z): 523 [M + H]⁺ Retention time (min): 4.37 (methodB) 23 (from IV and 1,2,3,4-tetrahydro- quinoline-N- carbonyl chloride)

15.4 MS-ESI pos. (m/z): 496 [M + H]⁺ Retention time (min): 4.45 (methodB)

24.4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl2-methoxyethylcarbamate

160.3 mg (0.29 mmol) of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl4-methoxybenzyl(2-methoxyethyl)carbamate from example IX are dissolvedin 3 ml of trifluoroacetic acid and stirred at 60° C. for 6 h. Forworkup, the reaction solution is poured into ice-water, cautiouslyneutralized with sodium bicarbonate and extracted 3× withdichloromethane, and the org. phase is dried over sodium sulfate andconcentrated. The residue is chromatographed on silica gel withdichloromethane/methanol (30:1).

Yield: 28.3 g (22.5%)

Rf: 0.88 (dichloromethane/methanol 10:1)

¹H-NMR: (200 MHz, CDCl3): δ=3.39 (s, 3H, OCH3), 3.43-3.58 (m, 4H,CH2—CH2), 5.15-5.28 (br. s, 2H, NH2), 5.50-5.63 (m, 1H, NH), 5.93 (s,2H, CH2), 6.86-7.30 (m, 5H, Ar—H), 8.37 (s, 1H, Ar—H), 8.59 (dd, 1H,Ar—H), 8.92 (dd, 1H, Ar—H).

MS: ESI pos.: m/z=438.2 [M+H]⁺

25.4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl1-piperazinecarboxylate

214.2 mg (0.390 mmol) of1-{4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl}4-tert-butyl 1,4-piperazinedicarboxylate from example 4 are dissolved in5 ml of dichloromethane, and 5 ml of trifluoroacetic acid are added. Themixture is left to stir at room temperature for 1 h. For workup, it isneutralized with 1 N sodium hydroxide solution and extracted three timeswith dichloromethane. The organic phase is dried over sodium sulfate andconcentrated in a rotary evaporator.

Yield: 123.1 mg (64.0%)

Rf: 0.16 (dichloromethane/methanol 10:1)

¹H-NMR: (200 MHz, D₆-DMSO): δ=2.67-2.81 (br.s, 4H, piperazine H),3.21-3.41 (br.s, 2H, piperazine H, coincident with H₂O signal),3.42-3.59 (br.s, 2H, piperazine H), 5.81 (s, 2H, CH₂), 7.08-7.41 (m, 7H,Ar—H and NH₂), 8.10 (s, 1H, pyrimidine H), 8.63 (dd, 1H, pyridine H),8.92 (dd, 1H, pyridine H)

MS: (ESI pos.), m/z=449.2 ([M+H]⁺)

26.4-{[(Diethylamino)carbonyl]amino}-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyldiethylcarbamate

100 mg (0.297 mmol) of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinolfrom example IV are suspended in 5 ml of anhydrous THF, and 7.51 mg(0.297 mmol) of sodium hydride (95%) are added. The mixture is left tostir at room temperature for 30 min. Then 44.35 mg (0.327 mmol) ofN,N-diethylcarbamoyl chloride are added, and the mixture is left to stirat room temperature overnight. Water is cautiously added, and themixture is extracted three times with ethyl acetate. The organic phaseis dried over, sodium sulfate and concentrated in a rotary evaporator.The residue is chromatographed on silica gel withdichloromethane/methanol 100:1 and then by preparative HPLC (column:Kromasil 100 C 18 5 μm 250×20 mm No. 101132R, flowrate: 25 ml/min, temp.50° C., water-acetonitrile 50/50).

Yield: 29.9 g (18.8%)

Rf: 0.88 (dichloromethane/methanol 10:1)

¹H-NMR: (400 MHz, D₆-DMSO): δ=1.07-1.19 (m, 12H,CH₃CH₂), 3.26-3.41 (m,8H, CH₃CH₂), 5.85 (s, 2H, CH₂), 7.11-7.44 (m, 5H, Ar—H), 8.52 (s, 1H,pyrimidine H), 8.68 (dd, 1H, pyridine H), 9.02 (dd, 1H, pyridine H),9.39 (br,s, 1H, NH)

27.4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinylmethylcarbamate

562.5 mg (1.04 mmol) of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl3,4-dimethoxybenzyl(methyl)carbamate from example VI are dissolved in 10ml of trifluoroacetic acid and stirred at 60° C. for 4 h. For workup,the reaction solution is poured into ice-water, cautiously neutralizedwith sodium bicarbonate and extracted three times with dichloromethane,and the organic phase is dried over sodium sulfate and concentrated. Theresidue is chromatographed on silica gel with dichloromethane/methanol(initially 100:1, then 36:1).

Yield: 123.8 g (30.4% over 2 stages)

MS(MALDI pos.): m/z=394.18 ([M+H]⁺), 416.15 ([M+Na]⁺)

Rf: 0.67 (dichloromethane/methanol 10:1)

¹H-NMR: (200 MHz, D₆-DMSO). The spectrum exhibits E/Z rotamerism of thecarbamate group. The following describes only the signals of the mainrotamer: δ=2.70 (d, 3H, CH₃), 5.81 (s, 2H, CH₂), 7.07-7.41 (m, 7H, Ar—Hand NH₂), 7.62 (br. q, 1H, NH), 8.11 (s, 1H, pyrimidine H), 8.62 (dd,1H, pyridine H), 8.93 (dd, 1H, pyridine H)

28.4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinylallyl(cyclopentyl)carbamate

100 mg (0.297 mmol) of4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinolfrom example IV are suspended in 15 ml of anhydrous pyridine and heatedto 110° C. 67.0 mg (0.357 mmol) of allyl(cyclopentyl)carbamoyl chloridefrom example VII are added to this in portions, and the mixture is leftto stir at 110° C. for 2 h. For workup, the reaction solution isconcentrated, the residue is taken up in dichloromethane and extractedonce with saturated ammonium chloride solution and once with saturatedNaCl solution, and the organic phase is dried over Na₂SO₄ andconcentrated in a rotary evaporator. The residue is chromatographed onsilica gel with dichloromethane/methanol 100:1.

Yield: 105 mg (72%)

Rf: 0.92 (dichloromethane/methanol 10:1)

¹H-NMR: (200 MHz, D₆-DMSO), δ=1.37-1.98 (br. m, 8H, cyclopentyl-CH₂),3.80-4.05 (br. m, 2H, allyl-CH₂N), 4.22-4.53 (br. m, 1H, cyclopentylCHN), 5.07-5.29 (br. m, 2H, 2H, olefinic allyl CH₂), 5.80-6.10 (br. m,1H, olefinic allyl CH), overlapped by 5.81 (s, 2H, CH₂), 7.04-7.41 (m,7H, Ar—H and NH₂), 8.08 (s, 1H, pyrimidine H), 8.64 (dd, 1H, pyridineH), 8.92 (dd, 2H, pyridine H)

MS: (ESI pos.), m/z=488 ([M+H]⁺), 975 ([2M+H]⁺)

29.N-[({4-Amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl}oxy)carbonyl]-N-(sec-butyl)glycine

73.5 mg (0.141 mmol) of ethylN-[({4-amino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl}oxy)carbonyl]-N-(sec-butyl)glycinatefrom example 20 are dissolved in 3 ml of THF/water/methanol 1:1:1. 5.1mg (0.12 mmol) of lithium hydroxide monohydrate are added to this, andthe mixture is left to stir at RT for 1 h. Workup involves extraction 3×with 10 ml of CH₂Cl₂ each time and 1× with saturated NaCl solution,drying over Na₂SO₄ and concentration to dryness in a rotary evaporator.

The residue is purified by preparative HPLC (column: Cromsil 120 ODS,C-18, 10 μm, 250×30 mm, flowrate 50 ml/min, room temp., gradient: wateracetonitrile at 0 min: 90:10, at 28 min 5:95).

Rf: 0.25 (CH₂Cl₂/CH₃OH 10:1)

HPLC retention time: 3.55 (method F)

MS-ESI pos.: (m/z): 494.3 [M+H]⁺, 987.3 [2M+H]⁺

1. A compound of the formula (I)

in which R¹ is hydrogen or a di-C₁₋₆-alkylaminocarbonyl radical, R² is aradical of the formula —O—CX—NR³R⁴, where X is O or S; R³ and R⁴ may beidentical or different from one another and each is a radical chosenfrom H, optionally substituted C₁₋₆-alkyl, optionally substitutedC₁₋₆-alkoxy-C₁₋₆-alkyl, optionally substituted hydroxy-C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substitutedC₁₋₆-alkylcarbonyloxy-C₁₋₆-alkyl, optionally substitutedC₁₋₆-alkoxycarbonyl-C₁₋₆-alkyl, optionally substitutedhydroxycarbonyl-C₁₋₆-alkyl, phenyl which is optionally substituted by aC₁₋₆-alkyl radical, a saturated five- to seven-membered heterocyclewhich is optionally linked via a C₁₋₆-alkyl radical to the nitrogenatom, and optionally substituted C₃₋₈-cycloalkyl, where R³ and R⁴ cannotsimultaneously be H; or R³ and R⁴ together with the nitrogen atom towhich they are bonded form a five- to seven-membered saturatedheterocycle which may optionally contain a further heteroatom chosenfrom N, O, and S and/or may optionally be substituted or fused to aphenyl ring; or a salt, stereoisomer, tautomer, or hydrate thereof. 2.The compound of claim 1, in which R¹ is hydrogen or adi-C₁₋₆-alkylaminocarbonyl radical, R² is a radical of the formula—O—CX—NR³R⁴, where X is O or S; R³ and R⁴ may be identical or differentand is a radical chosen from H, C₁₋₆-alkyl which optionally has a CN ora halogen substituent, C₁₋₆-alkoxy-C₁₋₆-alkyl, hydroxy-C₁₋₆-alkyl,C₂₋₆-alkenyl, C₁₋₆-alkylcarbonyloxy-C₁₋₆-alkyl,C₁₋₆-alkoxycarbonyl-C₁₋₆-alkyl, hydroxycarbonyl-C₁₋₆-alkyl, phenyl,p-tolyl, a saturated five- to seven-membered heterocycle which is linkedvia a C₁₋₆-alkanediyl radical to the nitrogen atom and has up to 2oxygen atoms, and optionally substituted C₃₋₈-cycloalkyl, where R³ andR⁴ cannot simultaneously be H; or R³ and R⁴ together with the nitrogenatom to which they are bonded form a five- or six-membered saturatedheterocycle which may optionally contain a further heteroatom chosenfrom N, O, and S and/or optionally an alkylcarbonyl or alkoxycarbonylsubstituent; or a salt, stereoisomer, tautomer, or hydrate thereof. 3.The compound of claim 1, in which R¹ is hydrogen or adiethylaminocarbonyl radical, R² is a radical of the formula—O—CX—NR³R⁴, where X is O or S; R³ and R⁴ may be identical or differentand each is a radical chosen from H, methyl, ethyl, isopropyl,butan-2-yl, methoxyethyl, 2-methoxy-1-methylethyl,1-cyano-1-methylethyl, 2-cyanoethyl, 2-chloroethyl,ethoxycarbonylmethyl, hydroxycarbonylmethyl 2-propenyl, phenyl, p-tolyl,1,3-dioxolan-2-methyl, cyclohexyl and cyclopentyl, where R³ and R⁴cannot simultaneously be H; or R³ and R⁴ together with the nitrogen atomto which they are bonded form a five- or six-membered saturatedheterocycle which may optionally contain a further heteroatom chosenfrom N and O and/or may optionally have a substituent chosen frommethylcarbonyl, ethoxycarbonyl and t-butoxycarbonyl, or together are1,2,3,4-tetrahydroquinolin-N-yl; or a salt, stereoisomer, tautomer, orhydrate thereof.
 4. A process for preparing compounds of formula (I),comprising reaction of a compound of the formula (II)

with a compound of the formula (III)

in an organic solvent in the presence of a base with heating andsubsequent conversion of the ether group into the free hydroxyl group togive a compound of the formula (IV)

and subsequent reaction of (IV) with a compound of the formulaX—CO—NR³R⁴ in which X is a halogen radical or alkoxy radical, and R³ andR⁴ have the meaning indicated in claim 1; in an organic solvent, whereappropriate in the presence of a base, and where appropriate withsubsequent removal of protective groups to give a compound of theformula (I).
 5. A pharmaceutical composition comprising at least onecompound of the formula (I) as defined in claim
 1. 6. A pharmaceuticalcomposition comprising at least one compound of the formula (I) asdefined in claim 1 in combination with at least one organic nitrate orNO donor.
 7. A pharmaceutical composition comprising at least onecompound of the formula (I) as defined in claim 1 in combination with atleast one compound which inhibits the breakdown of cyclic guanosinemonophosphate (cGMP).
 8. A method for the treatment of hypertension,comprising administering an effective amount of a compound of formula(I) as defined in claim
 1. 9. The method as claimed in claim 8, whereinthe compound of the formula (I) as defined in claim 1 is employed incombination with at least one organic nitrate or NO donor or incombination with at least one compound which inhibits the breakdown ofcyclic guanosine monophosphate (cGMP).